Brady Nathan R, Hamacher-Brady Anne, Yuan Hua, Gottlieb Roberta A
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA.
FEBS J. 2007 Jun;274(12):3184-97. doi: 10.1111/j.1742-4658.2007.05849.x.
Macroautophagy is a vital process in the cardiac myocyte: it plays a protective role in the response to ischemic injury, and chronic perturbation is causative in heart disease. Recent findings evidence a link between the apoptotic and autophagic pathways through the interaction of the antiapoptotic proteins Bcl-2 and Bcl-XL with Beclin 1. However, the nature of the interaction, either in promoting or blocking autophagy, remains unclear. Here, using a highly sensitive, macroautophagy-specific flux assay allowing for the distinction between enhanced autophagosome production and suppressed autophagosome degradation, we investigated the control of Beclin 1 and Bcl-2 on nutrient deprivation-activated macroautophagy. We found that in HL-1 cardiac myocytes the relationship between Beclin 1 and Bcl-2 is subtle: Beclin 1 mutant lacking the Bcl-2-binding domain significantly reduced autophagic activity, indicating that Beclin 1-mediated autophagy required an interaction with Bcl-2. Overexpression of Bcl-2 had no effect on the autophagic response to nutrient deprivation; however, targeting Bcl-2 to the sarco/endoplasmic reticulum (S/ER) significantly suppressed autophagy. The suppressive effect of S/ER-targeted Bcl-2 was in part due to the depletion of S/ER calcium stores. Intracellular scavenging of calcium by BAPTA-AM significantly blocked autophagy, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum calcium ATPase, reduced autophagic activity by approximately 50%. In cells expressing Bcl-2-ER, thapsigargin maximally reduced autophagic flux. Thus, our results demonstrate that Bcl-2 negatively regulated the autophagic response at the level of S/ER calcium content rather than via direct interaction with Beclin 1. Moreover, we identify calcium homeostasis as an essential component of the autophagic response to nutrient deprivation.
它在对缺血性损伤的反应中发挥保护作用，而慢性扰动是心脏病的病因。最近的研究结果表明，通过抗凋亡蛋白Bcl-2和Bcl-XL与Beclin 1的相互作用，凋亡途径和自噬途径之间存在联系。然而，这种相互作用促进还是阻断自噬的性质仍不清楚。在这里，我们使用一种高度敏感的、自噬特异性通量测定法，该方法能够区分自噬体产生增强和自噬体降解受抑制，研究了Beclin 1和Bcl-2对营养剥夺激活的巨自噬的控制。我们发现，在HL-1心肌细胞中，Beclin 1和Bcl-2之间的关系很微妙：缺乏Bcl-2结合结构域的Beclin 1突变体显著降低了自噬活性，这表明Beclin 1介导的自噬需要与Bcl-2相互作用。Bcl-2的过表达对营养剥夺的自噬反应没有影响；然而，将Bcl-2靶向肌浆网/内质网（S/ER）显著抑制了自噬。S/ER靶向的Bcl-2的抑制作用部分归因于S/ER钙储存的耗竭。BAPTA-AM对细胞内钙的清除显著阻断了自噬，而肌浆网/内质网钙ATP酶抑制剂毒胡萝卜素使自噬活性降低了约50%。在表达Bcl-2-ER的细胞中，毒胡萝卜素最大程度地降低了自噬通量。因此，我们的结果表明，Bcl-2在S/ER钙含量水平上负调节自噬反应，而不是通过与Beclin 1的直接相互作用。此外，我们确定钙稳态是对营养剥夺自噬反应的一个重要组成部分。
